Genomic characterization of Aeromonas spp. isolates from striped catfish with motile Aeromonas septicemia and human bloodstream infections in Vietnam

Abstract Aeromonas spp. are commonly found in the aquatic environment and have been responsible for motile Aeromonas septicemia (MAS) in striped catfish, resulting in significant economic loss. These organisms also cause a range of opportunistic infections in humans with compromised immune systems. Here, we conducted a genomic investigation of 87 Aeromonas isolates derived from diseased catfish, healthy catfish and environmental water in catfish farms affected by MAS outbreaks in eight provinces in Mekong Delta (years: 2012–2022), together with 25 isolates from humans with bloodstream infections (years: 2010–2020). Genomics-based typing method precisely delineated Aeromonas species while traditional methods such as aerA PCR and MALDI-TOF were unable identify A. dhakensis. A. dhakensis was found to be more prevalent than A. hydrophila in both diseased catfish and human infections. A. dhakensis sequence type (ST) 656 followed by A. hydrophila ST251 were the predominant virulent species-lineages in diseased catfish (43.7 and 20.7 %, respectively), while diverse STs were found in humans with bloodstream infections. There was evidence of widespread transmission of ST656 and ST251 on striped catfish in the Mekong Delta region. ST656 and ST251 isolates carried a significantly higher number of acquired antimicrobial resistance (AMR) genes and virulence factors in comparison to other STs. They, however, exhibited several distinctions in key virulence factors (i.e. lack of type IV pili and enterotoxin ast in A. dhakensis), AMR genes (i.e. presence of imiH carbapenemase in A. dhakensis), and accessory gene content. To uncover potential conserved proteins of Aeromonas spp. for vaccine development, pangenome analysis has unveiled 2202 core genes between ST656 and ST251, of which 78 proteins were in either outer membrane or extracellular proteins. Our study represents one of the first genomic investigations of the species distribution, genetic landscape, and epidemiology of Aeromonas in diseased catfish and human infections in Vietnam. The emergence of antimicrobial resistant and virulent A. dhakensis strains underscores the needs of enhanced genomic surveillance and strengthening vaccine research and development in preventing Aeromonas diseases in catfish and humans, and the search for potential vaccine candidates could focus on Aeromonas core genes encoded for membrane and secreted proteins.


Abstract
Aeromonas spp.are commonly found in the aquatic environment and have been responsible for motile Aeromonas septicemia (MAS) in striped catfish, resulting in significant economic loss.These organisms also cause a range of opportunistic infections in humans with compromised immune systems.Here, we conducted a genomic investigation of 87 Aeromonas isolates derived from diseased catfish, healthy catfish and environmental water in catfish farms affected by MAS outbreaks in eight provinces in Mekong Delta (years: 2012-2022), together with 25 isolates from humans with bloodstream infections (years: 2010-2020).Genomics-based typing method precisely delineated Aeromonas species while traditional methods such as aerA PCR and MALDI-TOF were unable identify A. dhakensis.A. dhakensis was found to be more prevalent than A. hydrophila in both diseased catfish and human infections.A. dhakensis sequence type (ST) 656 followed by A. hydrophila ST251 were the predominant virulent species-lineages in diseased catfish (43.7 and 20.7 %, respectively), while diverse STs were found in humans with bloodstream infections.There was evidence of widespread transmission of ST656 and ST251 on striped catfish in the Mekong Delta region.ST656 and ST251 isolates carried a significantly higher number of acquired antimicrobial resistance (AMR) genes and virulence factors in comparison to other STs.They, however, exhibited several distinctions in key virulence factors (i.e.lack of type IV pili and enterotoxin ast in A. dhakensis), AMR genes (i.e.presence of imiH carbapenemase in A. dhakensis), and accessory gene content.To uncover potential conserved proteins of Aeromonas spp.for vaccine development, pangenome analysis has unveiled 2202 core genes between ST656 and ST251, of which 78 proteins were in either outer membrane or extracellular proteins.Our study represents one of the first genomic investigations of the species distribution, genetic landscape, and epidemiology of Aeromonas in diseased catfish and human infections in Vietnam.The emergence of antimicrobial resistant and virulent A. dhakensis strains underscores the needs of enhanced genomic surveillance and strengthening vaccine research and development in preventing Aeromonas diseases in catfish and humans, and the search for potential vaccine candidates could focus on Aeromonas core genes encoded for membrane and secreted proteins.

Impact Statement
Aeromonas spp.are opportunistic pathogens in the aquatic environment and have been responsible for motile Aeromonas septicemia (MAS) in striped catfish farms along Vietnam's Mekong Delta region.There are two prevalent MAS-causing Aeromonas species, namely A. hydrophila and A. dhakensis, which have been characterized by the presence of diverse virulence and antibiotic resistance traits.Even though epidemiological investigations of Aeromonas infections are urgently needed; such efforts were hindered by the lack of reliable species identification methods.Here, by using whole genome sequencing data we uncovered the predominance of A. dhakensis ST656 and A. hydrophila ST251 in diseased catfish and their geographical spread in the Mekong Delta region.These two species-lineages were undetected in human-derived isolates; instead, human bloodstream infections were caused by a diverse array of sequence types.Compared to A. hydrophila, the gene clusters encoding type IV pili and a heat-stable enterotoxin were missing from most of the A. dhakensis isolates.Generally, A. hydrophila and A. dhakensis isolates from diseased catfish contained more antimicrobial resistance genes and virulence factors compared to humanderived isolates.Our study provides new insights into the species distribution, highlighting the emergence of virulent and antimicrobial resistance lineage ST656 of A. dhakensis in Vietnam.We also revealed the distinctions in genotype distribution, virulence factors and AMR gene content between A. dhakensis ST656 and A. hydrophila ST251, as well as between aquaculturederived and human-derived isolates.Our research will pave the way for further research in molecular surveillance and vaccine development to understand the transmission of Aeromonas in a broader context and prevent the MAS in catfish.

INTRODUCTION
Vietnam has been a significant producer of aquaculture products, notably striped catfish (Pangasianodon hypophthalmus), which is exported worldwide under the name pangasius [1].The production of striped catfish is mainly concentrated in the Mekong Delta region and has a pivotal role in the regional economy with exports worth more than USD 1.7 billion [2].However, outbreak of bacterial diseases, such as motile Aeromonas septicemia (MAS) caused by Aeromonas hydrophila or bacillary necrosis of pangasius (BNP) caused by Edwardsiella ictaluri can result in high mortality rates in striped catfish and have a detrimental impact on fish production [3].
Antibiotics are commonly used to treat diseased catfish during outbreak and decontaminate the water in the fish ponds [4].However, antibiotic use can promote the development of antimicrobial resistance (AMR) in both environmental and the infecting organisms, primarily through the horizontal transfer of AMR plasmids [5,6].The circulation of multidrugresistant (MDR) A. hydrophila and A. dhakensis strains has been reported in fish farms in Northern and Southern Vietnam.These strains carried multiple AMR genes conferring resistance against various antibiotics, including oxacillin, amoxicillin, vancomycin, erythromycin, oxytetracycline, florfenicol, and sulfamethoxazole [7].Consequently, there is a crucial need for developing inexpensive vaccines that are straightforward to deliver for preventing the diseases caused by Aeromonas in catfish and limiting the use of antibiotics.
The importance of vaccine development is further emphasized in the context of the emergence of hypervirulent Aeromonas (vAh) strains.Recent studies have shown the appearance of a vAH, namely sequence type (ST) 251, posing a serious concern in aquaculture globally.These strains have been responsible for several MAS outbreaks in farmed fish in the USA and China [8,9].Moreover, isolates within this lineage have recently been detected in provinces farming striped catfish in Vietnam [10,11].The vAh lineage possesses a diverse array of virulent factors and multiple AMR genes.Furthermore, A. dhakensis strains that display virulence in both catfish and humans have also emerged recently.A. dhakensis share a high nucleotide identity to A. hydrophila and thus was misclassified as Aeromonas hydrophila subsp.dhakensis [12].Since 2013, A. dhakensis was formally acknowledged as a distinct Aeromonas species [13], encompassing what were previously classified as Aeromonas hydrophila subsp.dhakensis and A. aquariorum [14,15].In the Mekong Delta region of Vietnam, the first report of diseased striped catfish caused by A. dhakensis was linked to the genotype ST656 [16].
Aeromonas spp.can cause life-threatening invasive infections in humans, particularly immunocompromised individuals.There has been very few research on human Aeromonas infections in Vietnam and the extent of genetic and species overlap to those present in non-human sources.Furthermore, traditional method such as biochemical testing, protein-based MALDI-TOF or gene-based 16S rRNA typing often fail to distinguish closely related Aeromonas species, hindering a comprehensive understanding of the population structure and transmission patterns of these organisms [17].In this investigation, we utilized whole genome sequencing approach to precisely determine Aeromonas species, characterize the gene content and depict the genomic epidemiology of Aeromonas isolates derived from diseased striped catfish and human infections in Southern Vietnam.

Sampling sites and primary isolation of Aeromonas spp. isolates
Eighty-seven Aeromonas spp.strains were collected in different fish farms in the Mekong Delta region between 2012 and 2022, encompassing several provinces: An Giang, Vinh Long, Dong Thap, Tien Giang, Can Tho, Ben Tre.The sources of bacterial isolation included the kidneys or livers of diseased catfish, encompassing juvenile, fingerling, and grown-up specimens (n=63), as well as healthy catfish (n=12) from the same farms.Additionally, 12 water samples were also collected from the farms housing the diseased catfish.Samples were grown on Trypticase soy broth (TSB) or Brain Heart Infusion (BHI) broth overnight and suspected colonies were identified by API 20E biochemical testing, and the presence of MAS-causing Aeromonas was confirmed by PCR assay targeting the 16S rRNA and a 209 bp region of the aerolysin-encoding aerA gene [18].Among 87 isolates derived from catfish and water samples, 31 isolates were further classified as either A. hydrophila or A. dhakensis by rpoD sequencing method [19].Additionally, 25 A. hydrophila isolates (classified by MALDI-TOF) identified from patients with bloodstream infections at Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam were included, giving rise to a total of 112 Aeromonas isolates.

DNA extraction and whole genome sequencing
DNA was extracted from the overnight culture of 112 Aeromonas isolates using the Wizard Genomic DNA Extraction Kit (Promega, USA) following the manufacturer's recommendations.DNA library preparation was done using a Nextera XT kit, followed by genome sequencing on an Illumina Miseq or NextSeq platform (Illumina, USA) to generate either 150 bp (for human-related isolates) or 75 bp paired-end reads (for catfish-and water-related isolates).Illumina reads quality assessment were performed by FASTQC [20] and de novo assembled using Unicycler v0.4.8 with default parameters [21].The raw Illumina sequencing reads of each isolate have been deposited in ENA with the project accession PRJEB65955 (Table S2).

Phylogenetic tree and data visualization
Based on the concatenated sequences of the six housekeeping genes from the MLST scheme (gyrB, groL, gltA, metG, ppsA, and recA), single nucleotide polymorphisms (SNP) were extracted using SNP-sites v2.5.1 [31], yielding a total of 203 SNPs (for A. hydrophila) and 306 SNPs (for A. dhakensis).Phylogenetic tree was reconstructed based on the resulting SNP alignment of each species, using maximum likelihood (ML) method and best-fit substitution model implemented in IQ-TREE v2.2.2.4 [32].The ML tree was visualized and annotated using iTOL [33].

Statistical analysis
T-test was used to analyse the differences in the number of virulence factors between A. hydrophila and A. dhakensis.P < 0.05 was considered to be statistically significant.The analysis was performed using the built-in ttest_ind function from the Scipy library written in Python programming language.
We compared the genomics-based ANI method with rpoD sequencing, aerA PCR and MALDI-TOF for the identification of Aeromonas species, using the available data.Compared to the ANI method, MALDI-TOF and aerA PCR had the lowest agreement rates of 12 % (3/25) and 18.6 % (11/59), respectively; moreover, they misclassified A. dhakensis and other Aeromonas spp. as A. hydrophila (Fig. 1b).The rpoD sequencing method exhibited a high agreement rate with the ANI method (89.7 %, 26/29).However, it misidentified two A. hydrophila isolates as A. dhakensis and one Aeromonas veronii isolate as A. hydrophila.

Distribution of virulence factors in A. dhakensis and A. hydrophila isolates
Here, we aimed to characterise and compare the distribution of key virulence factors between A. dhakensis and A. hydrophila isolates (Table 2).Among aquaculture-related isolates, the Type IV Flp, Msh, and Tap pili, which play a role in adherence and biofilm formation, were either missing (Flp and Msh) or incomplete (Tap with tapB gene missing) in A. dhakensis isolates.They were however detected in 78-100 % of A. hydrophila isolates.All A. dhakensis and A. hydrophila isolates harboured lateral flagella.The A. dhakensis isolates did not contain the genes responsible for polar flagella, whereas the A. hydrophila isolates possessed the flagellin-encoding flaAB genes but lacked the accessory factor modification gene maf-1.Notably, the distribution of secretion systems, toxins, and enzymes in aquaculture-related A. hydrophila and A. dhakensis isolates exhibited high similarities.The cytotoxic enterotoxin-encoding AerA gene was identified in 87 % (61/70) of Aeromonas isolates, while the hemolysin-encoding genes ahh1 and hlyA were detected in all isolates.Intriguingly, 91.3 % (21/23) of A. hydrophila isolates possessed the heat-stable cytotonic enterotoxin ast, while only 2.1 % (1/47) of A. dhakensis carried this virulent factor.Meanwhile, the key enzymes such as protease, elastase, lipase, and nuclease were present in nearly all isolates.Among BSI-related isolates, both Aeromonas showed an absence of factors contributing to adhesion, with the only exception belonged to Msh pili in 33.3 % of A. hydrophila (1/3) and Tap pili in 20 % of A. dhakensis (4/20).Virulence factors involved with polar flagellum were undetected in both Aeromonas, while only A. hydrophila shown the presence of lateral flagella.Intriguingly, both aerolysin (aerA) and rtx were absent from human-related A. hydrophila but were detected in 35 % (7/20) and 85 % (17/20) of A. dhakensis, respectively.It is also noteworthy that most degradative enzymes did not present in both BSI-related Aeromonas, with only Exu1 being detected in 33.3 % (1/3) of A. hydrophila and EprA1 in 5 % (1/20) of A. dhakensis.Both secretion systems (T2SS and T6SS) were also detected in both Aeromonas with different distributions.Overall, the A. dhakensis and A. hydrophila isolates derived from aquaculture samples carried a significantly higher number of virulence factors in comparison to that of human BSI isolates (131.0 vs. 94.0,t-test two-tails, P=1.7×10 −16 , Table S3).

Phylogenetic structure of A. hydrophila isolates
We sought to understand the genetic relatedness and transmission of A. hydrophila isolates as well as the distribution of AMR genes and virulence factors across different sequence types (STs).Therefore, a phylogenetic tree was reconstructed encompassing 23 isolates sourced from diseased catfish and water, and three isolates from human bloodstream infections (Table S1).All the ST251 isolates from infected fish and water samples formed a separate cluster on the phylogenetic tree, exhibiting limited genetic diversity (mean pairwise SNP distance of 37 SNPs), as shown in Fig. 2.These isolates were identified between 2012 and 2022 and were distributed across multiple provinces in the Mekong Delta region, indicating sustained and widespread transmission of this lineage.Overall, the non-ST251 isolates were distantly related to ST251 and displayed a higher degree of genetic diversity.ST251 isolates carried a higher abundance of virulence factors in comparison to other STs.More specifically, non-ST251 isolates were characterised by the absence of both flaAB and tapA genes, encoding for the polar flagella and the type IV tap pili, respectively (Fig. 2).Compared to the aquaculture-related isolates, isolates derived from human BSIs lacked a number of virulence factors, including the lateral flagella (flgA-N), T6SS (vgrG, vasH/vasK), protease (eprA1), elastase (ahpB), nuclease (exu1), lipase (lip) and repeat in toxin (RTX toxin).We observed that the aerolysin-encoding aerA gene was not found in seven out of eight non-ST251 isolates, providing further indication that aerolysin is not a suitable marker for the identification of A. hydrophila.Meanwhile, a novel ST (STNF: gyrB-380, groL-710, metG-842, ppsA-780) was detected in two isolates from diseased catfish between 2015 and 2016, characterised by possessing more virulence factors compared to other non-ST251.However, compared to ST251, they still lacked flaAB, tapABCD, aerA, and lip genes.

Phylogenetic of A. dhakensis isolates
The phylogenetic tree of A. dhakensis isolates showed that the ST656 isolates from diseased catfish and water formed a separate lineage, with limited genetic variation (mean pairwise SNP distance of 71 SNPs) (Table S1, Fig. 3).Akin to A. hydrophila ST251, A. dhakensis ST656 appeared to spread across multiple provinces in the Mekong Delta.Conversely, the non-ST656 isolates exhibited a greater level of diversity, with 21/29 isolates belonging to novel STs (STNF), each exhibiting distinct allelic combinations.The isolates obtained from human BSIs were genetically distantly related.Again, we observed that the aquaculture-related isolates carried a significantly higher number of virulence factors compared to those derived from human BSIs (P<3.0×10−9 ).For example, the corresponding prevalence of intact flagella, T2SS, T6SS, toxins and enzymes in BSI-derived isolates were 0, 34.8, 8.7, and 62.4 %, compared to 62.9, 100, 88.6, and 93.4 % in aquaculture-related isolates.
Regarding the AMR gene profile, most A. dhakensis strains harboured a combination of the carbapenem resistance gene imiH (94.0 %) and a chromosomal class C β-lactamase bla AQU gene (98.5 %) conferring resistance to cefotaxime.Compared to other STs, ST656 isolates possessed more AMR genes.Apart from the imiH-bla AQU genes which were present in all ST656 isolates, they also carried different combinations with other AMR genes such as qnrS2, floR, sul, tetA and dfrA (Fig. 3).

Pangenome analysis and localization prediction of core genes within A. hydrophila ST251 and A. dhakensis ST656
A pangenome analysis was performed to investigate the genetic landscape and functional diversity between the two major species-lineages of A. hydrophila-ST251 and A. dhakensis-ST656 (Fig. 4).Out of 56 genomes of ST251 (n=18) and ST656 (n=38), the total number of genes found were 6782, of which 2202 genes were part of core genome (present in 99 % of the isolates).Of the 4580 accessory genes, 3306 were identified as shell genes (present in >15 % and <95 % of the isolates) and 874 were characterized as cloud genes (present in 15 % or less of the isolates).
To determine the cellular location of core genes, cellular localization prediction was performed by CELLO2GO on 2202 core genes, in which predicted outer membrane and extracellular proteins were further confirmed by DeepTMHMM.To this end, there were 18 proteins in the extracellular regions, 60 outer membrane proteins, 270 periplasmic proteins, 468 inner membrane proteins, and 1386 cytoplasmic proteins (Table S4).Further functional annotation of extracellular and outer membrane proteins by searching the Clusters of Orthologous Groups (COG) showed that 28.2 % (22/78) was contributing to cell wall/membrane/envelope biogenesis, 23.1 % (18/78) was related to transport pathways, 19.2 % (15/78) was related to cell motility, while proteins contributing to the post-translational modification or cell division only accounted for 6.4 % of the subset.Nevertheless, 21.8 % of the extracellular/outer membrane proteins were still of unknown function, which required more research to accurately annotate.

DISCUSSION
In this study, by comparing genomics-based ANI with other typing methods in the identification of Aeromonas species, we found the inaccuracy of gene-based typing (aerA PCR) or protein-based method (MALDI-TOF) in the detection of A. dhakensis.The lack of discriminatory power of these traditional methods might have resulted in a common perception that A. hydrophila is the primary causative agent of diseases in fish and humans [34].In fact, our study findings revealed that A. dhakensis is a dominant cause of infections in both catfish (77 %) and bloodstream infections in humans (80 %).This result aligned with previous study showing the emergence of A. dhakensis in human infections [17,35] and in catfish from Vietnam [16,34].However, our study also uncovered the presence of other Aeromonas species presented in the aquaculture-related samples: A. veronii, A. caviae, A. salmonicida and A. enteropelogenes.Among these, A. veronii, an emerging human enteric pathogen [36], was identified in both aquaculture and human BSI.Our research underscores the importance of developing novel markers, updating the existing MALDI-TOF database, or employing sequencing-based methods for a more comprehensive characterization of these species as well as their respective epidemiological and genetic profiles.Furthermore, research on vaccine and therapeutics may consider shifting their focus toward A. dhakensis given its significance and genomic dissimilarity from A. hydrophila.
Aeromonas are widely distributed in aquatic environment and can cause severe bacteremia in immunocompromised humans with a high mortality rate [37].Our study represents the first genomic investigation of Aeromonas isolates causing bloodstream infections in humans in Vietnam.The source of Aeromonas bacteremia may involve ingestion of contaminated food or water followed by intestinal colonization and gut translocation in people with weakened immune system [38].Here, human Aeromonas bacteremia is attributed to a wide range of sequence types (STs), in contrast to motile Aeromonas septicemia (MAS) in catfish, which is primarily caused by A. hydrophila-ST251 and A. dhakensis-ST656.While the presence of novel ST in human Aeromonas spp. was well within expectations [39], the presence of new STs in Vietnam aquaculture samples was unprecedented.Aeromonas isolates from humans with BSIs also distinct AMR and virulence gene profiles compared to the aquaculture-related isolates.These findings indicate that catfish exposure or catfish consumption is unlikely to be a source of human infections.
The A. hydrophila and A. dhakensis isolates derived from diseased catfish and water possessed a remarkably high number of virulence factors, encompassing motility, adhesion, secretion systems, various toxins and enzymes.Conversely, many virulence factors such as lateral flagella, T6SS, degradative enzymes (protease, elastase, nuclease, lipase) and toxin (RTX toxin) were either missing or present with significantly lower prevalence in human BSI-derived isolates.This finding implies that the virulence factors that are crucial for causing diseases in catfish may not necessarily have the same significance in human disease.Furthermore, there was an inherent difference between A. hydrophila and A. dhakensis regarding genes contributing to adhesions.Specifically, genes involving in the biogenesis and function of msh, flp and tap type IV pilus was absent in A. dhakensis, which further supports the notion that type IV pili might not be fundamental for the pathogenesis of Aeromonas [40].Furthermore, the heat-stable cytotonic enterotoxin Ast, which was linked to clinical gastroenteritis [41] and increased fluid secretion in mouse model [42] was not detected in 98 % of A. dhakensis.Collectively, we reckon, the pathogenesis of A. hydrophila and A. dhakensis infections may have distinct features, warranting more in-depth investigations.We also observed that the two dominant-genotypes of A. hydrophila-ST251 and A. dhakensis-ST656 contained more virulence factors than other aquaculture-related isolates.For example, the mobility and adhesion factors were missing in non-ST251 A. hydrophila, and the aerA hemolysin was absent in non-ST251 A. hydrophila and non-ST656 A. dhakensis isolates.We speculate that the two prevalent genotypes of ST251 and ST656 are the main culprits for MAS in catfish, while other genotypes coexist in the same ecological niches.

Fig. 1 .
Fig. 1.Comparison of different methods for species identification of Aeromonas.(a) The distribution of ANI values (represented in boxplot) between assembled genomes and each of the reference genomes.The dash line shows the ANI cut-off value of 96% for species identification.An ANI value above 96% indicates that the isolate belongs to the same species as the reference genome.(b) Statistics of agreement between rpoD sequencing, aerA PCR and MALDI-TOF in species identification.Colored cells indicate disagreements between non-ANI methods and ANI method.

1 .
The distribution of sequence type (STs) within Aeromonas isolates

Fig. 2 .
Fig. 2. Maximum likelihood phylogenetic tree of A. hydrophila isolates from fish, water and bloodstream infections The node is coloured based on the sequence types (STs) of isolates.The first and second bar show the year and location of sample collection, respectively.The first heatmap illustrates the presence of acquired AMR genes.The second heatmap depicts the presence of key virulence factors.The tree scale represents the number of substitutions per site.

Fig. 3 .
Fig.3.Maximum likelihood phylogenetic tree of A. dhakensis isolates from fish, water and bloodstream infections The node is coloured based on the sequence types (STs) of isolates.The first and second bar show the year and location of sample collection, respectively.The first heatmap illustrates the presence of acquired AMR genes.The second heatmap depicts the presence of key virulence factors.The tree scale represents the number of substitutions per site.

Table 2 .
Distribution of key virulence factors in A. hydrophila and A. dhakensis isolates